1. An AP2 Transcription Factor Is Required for a Sleep-Active Neuron to Induce Sleep-like Quiescence in C. elegans 
Michal Turek, Ines Lewandrowski, Henrik Bringmann 
Current Biology 
Volume 23, Issue 22, Pages (November 2013)
DOI: /j.cub
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2. Current Biology 2013 23, 2215-2223DOI: (10.1016/j.cub.2013.09.028)
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3. Figure 1 aptf-1 Is Required for Behavioral Quiescence
Error bars are SEM.
(A and B) Nose speed during sleep-like and wake-like behavior in an individual wild-type (A) and aptf-1 mutant (B). The red arrow indicates the shedding of the cuticle.
(C–E) Probability distribution of nose speeds during wake-like and sleep-like behavior in wild-type (C), aptf-1(gk795) (D), and aptf-1(tm3287) (E).
(F–I) aptf-1 rescue experiments. Probability distribution of nose speeds using endogenous promoter expression (F), AIB expression (G), RIB expression (H), and RIS expression (I) are shown. Nose speed during the wake-like feeding period is displayed in red; nose speed during the nonfeeding sleep-like period is displayed in blue. The wake-like period used was 2 hr before the onset of sleep-like behavior.
(J and K) aptf-1 mutants lack shutdown of command interneuron activity. Shown are AVA/AVE calcium transient frequency (J) and intensity (K) for wild-type and aptf-1 mutants. ∗∗ denotes statistical significance with p < 0.01, Wilcoxon signed-rank test.
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4. Figure 2
aptf-1 Expresses Strongly in Only Five Interneurons, AIB, RIB, and RIS
Error bars are SEM.
(A) Spinning-disk and differential interference contrast images of transgenic animals expressing a destabilized version of GFP under the control of the aptf-1 promoter show that aptf-1 is expressed in AIB, RIB, and RIS. Scale bar represents 10 μm.
(B) aptf-1 limits its own expression. Expression quantification from embryo until adult stage showed that aptf-1 promoter activity was strongest during embryogenesis and early larval development and decreased until the adult stage. In aptf-1 mutants, promoter activity did not decrease during larval development indicating that aptf-1 controlled its own expression.
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5. Figure 3
The AIB and RIB Neurons Are Active during Wake-like Behavior, and RIS Is Most Active at the Beginning of Sleep-like Behavior
Error bars are SEM.
(A) Calcium transient frequency and intensity in AIB showed reduced activity in this neuron during sleep-like behavior that depended on aptf-1.
(B) Calcium transient frequency and intensity in RIB showed reduced activity during sleep-like behavior that depended on aptf-1.
(C) Averaged calcium activity of RIS across time showed that RIS was most active during the transition from wake-like (red) to sleep-like (blue) behavior. RIS still activated at the onset of the sleep-like period in aptf-1 mutant worms.
(D) Manipulation of the behavioral state also changed RIS activity. When RIS activity was high and the worms were immobile, strong mechanical stimulation caused a reversal of the sleep-like behavior and an acute inhibition of RIS. When RIS activity was low and the worms were immobile, stimulation caused a reversal of sleep-like behavior and an activation of RIS activity that coincided with a restoration of quiescence. The stimulation period is shown in gray. ∗ denotes statistical significance with p < 0.05, ∗∗∗ denotes statistical significance with p < 0.001, Wilcoxon signed-rank test.
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6. Figure 4
Activating aptf-1-Expressing Neurons during Wake-like Behavior Causes Reversible Behavioral Quiescence
Error bars are SEM.
(A) Channelrhodopsin activation of aptf-1-expressing neurons during wake-like behavior caused acute immobility.
(B) This immobility depended on aptf-1, because blue-light stimulation in aptf-1 mutants did not cause quiescence but caused an increase in movement instead.
(C and D) Control experiment without all-trans retinal (ATR) for wild-type (C) and aptf-1 mutant worms (D).
(E and F) Quiescence caused by channelrhodopsin-based activation of aptf-1-expressing neurons is reversible. A dish-tapping stimulus (dark gray) delivered during ChR2 activation (light gray) causes resumption of movement (E). Control experiment without ATR is shown in (F). ∗∗∗ denotes statistical significance with p < 0.001, Wilcoxon signed-rank test.
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7. Figure 5
Ablation of a Single Neuron, RIS, Strongly Impairs Behavioral Quiescence
Probability distributions of nose speed during wake-like and sleep-like behavior are shown. Wilcoxon signed-rank test was used.
(A) Mock-ablated worms.
(B) RIS ablation.
(C) AIB ablation.
(D) RIB ablation.
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8. Figure 6
Neuropeptide Signaling, but Not GABA, Is Essential for Sleep-like Quiescence
Error bars are SEM.
(A–C) The GABA null mutant unc-25(e156) shows quiescence.
(A) Probability distribution of nose speeds.
(B) Induction of quiescence by channelrhodopsin-based activation of aptf-1-expressing neurons.
(C) Control without ATR.
(D–F) The neuropeptide proprotein convertase mutant egl-3(gk238) shows quiescence defects. Wilcoxon signed-rank test was used.
(D) Probability distribution of nose speeds shows a strong reduction of quiescence.
(E) No induction of quiescence by channelrhodopsin-based activation of aptf-1-expressing neurons
(F) Control without ATR.
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